The present invention relates to methods of constructing and screening a library of polynucleotide sequences of interest in filamentous fungal cells.
Filamentous fungi have been widely used as host cells for the commercial production of polypeptides. However, when it is desirable to produce a variant of the polypeptide with specified altered characteristics, e.g., thermostability, pH activity profile, specific activity, substrate specificity, Km, Vmax etc., the construction and screening of a library of variant encoding sequences commonly requires the use of an intermediate host, e.g., bacterial cells or yeast, due to the low frequency of transformation and a variation in copy number among independently transformed filamentous fungal cells.
Several methods for the construction of libraries of polynucleotide sequences of interest in yeast have been disclosed in which the libraries are screened in yeast prior to transformation of a production relevant host, such as, for example, filamentous fungi with the potential variant polynucleotide sequences of interest.
Often, however, a polynucteotide sequence identified by screening in yeast or bacteria cannot be expressed or is expressed at low levels when transformed into production relevant filamentous fungal cells. This may be due to any number of reasons, including differences in codon usage, regulation of mRNA levels, translocation apparatus, post-translational modification machinery (e.g., cysteine bridges, glycosylation and acylation patterns), etc.
Secondly, whether a polynucleotide sequence of interest would be expressed in the production host at commercially useful levels is not necessarily predictable. For example, if the organism used in screening the library is, e.g., a bacterial or yeast cell and the production relevant host cell is a filamentous fungal cell, the protease profiles differ. Thus, a sequence encoding one or more characteristics of interest which has been identified in yeast may be degraded by proteases expressed in the product relevant filamentous fungal host cell. Furthermore, to obtain optimized yields of the expressed product by altering the function of regulatory proteins or regulatory sequences requires direct manipulation of the production host.
A. Aleksenko and A. J. Clutterbuck (1997. Fungal Genetics and Biology 21:373-387) disclose the use of autonomous replicative vectors, or autonomously replicating sequences (ARS), for gene cloning and expression studies. AMA1 (autonomous maintenance in Aspergillus) is one of the plasmid replicator elements discussed. It consists of two inverted copies of a genomic repeat designated MATE1 (mobile Aspergillus transformation enhancer) separated by a 0.3 kb central spacer. AMA1 promotes plasmid replication without rearrangement, multimerization or chromosomal integration.
It has been found that AMA1-based plasmids provide two advantages in gene cloning in filamentous fungi. The first is a high frequency of transformation which both increases the potential library size and can eliminate the need for library amplification in an intermediate host, e.g., E. coli, so that a recipient Aspergillus strain can be transformed directly with a ligation mixture. Secondly, by providing a stable and standard environment for gene expression, the properties of the transformants will be uniform.
It is an objective of the present invention to provide improved methods for constructing and screening libraries of polynucleotide sequences of interest in filamentous fungal cells by use of an episomal replicating DNA vector to provide a high frequency of transformation and a uniformly high level of gene expression among independently transformed cells. By minimizing variation in copy number among independently transformed cells, a variant polypeptide of interest may be identified directly on the basis of expression of the characteristics of interest.
Accordingly, in a first aspect the present invention relates to a method of constructing and selecting or screening a library of polynucleotide sequences of interest in filamentous fungal cells wherein the method comprises:
(a) transforming the fungal cells with a population of DNA vectors, wherein each vector comprises:
(i) a polynucleotide sequence encoding a fungal selective marker and a fungal replication initiating sequence, wherein the marker and the replication initiating sequence do not vary within the population; and
(ii) a polynucleotide sequence of interest wherein the population of DNA vectors contains more than one variant of the polynucleotide sequence;
(b) cultivating the cells under selective pressure;
(c) selecting or screening for one or more transformants expressing a desired characteristic; and
(d) isolating the transformant(s) of interest.
In other aspects, the invention relates to the use of a fungal replication initiating sequence to construct a library of polynucleotide sequences of interest and to screen or select a library of such polynucleotide sequences.